Androgen Deprivation Therapy (ADT) has been the standard-of-care for advanced prostate cancer for almost 70 years, but is neither curative nor durable. Furthermore, ADT is not an option for treatment of organ-localized prostate cancer because of collateral systemic morbidity. The hypothesis of this project is ... interdiction of prostate endothelial cell-specific uptake and trans-cellular transport of circulating adrenal androgens through the blood-prostate barrier, will complement ADT to provide a curative/durable therapy for organ-localized and advanced prostate cancer. The experimental goals of this project are to determine if dehydroepiandrosterone-sulfate (DHEA-S) represents an endogenous substrate for intracrine dihydrotestosterone (DHT) production and AR-activation by prostate epithelial cells in both the presence and absence of circulating testosterone (ADT), if intracrine metabolism of DHEA-S?DHT is mediated through a steroid 5?-reductase 1 (SRD5A1)- or a SRD5A2-dependent mechanism, and if prostate endothelial cell- specific mechanisms of DHEA-S uptake, transport or efflux represent prostate endothelial cell-specific therapeutic targets to make ADT more effective. Aim 1 will determine inter-patient variability in up-take and metabolism of circulating testosterone and DHEA-S, the expression profiles of genes associated with androgen metabolism in prostate endothelial cells and prostate cancer/prostate epithelial cells, the short-term effect of testosterone-deprivation on these processes, and whether intracrine metabolism of DHEA-S ? DHT is mediated by SRD5A1 or SRD5A2. Aim 2 will define the molecular mechanisms that mediate uptake, trans-cellular transport and efflux of DHEA-S in human prostate endothelial cells and prostate cancer/prostate epithelial cells, and identify the metabolites effluxed by prostate endothelial cells that serve as precursors for intracrine conversion to DHT in prostate cancer/prostate epithelial cells. Aim 3 wil determine whether interdiction of adrenal androgen uptake/metabolism/efflux by prostate endothelial cells and/or prostate cancer/prostate epithelial cells using inhibitors of SLCO/SLC uptake transporters, ABC efflux pumps, STS and SRD5A1&2 enhances the ability of testosterone-deprivation (ADT) to block intracrine production of DHT. Aims 1 and 2 will be conducted in ex vivo and in vitro model systems using small pieces of intact freshly procured prostate tissue and primary cultures of prostate endothelial cells and prostate cancer cells. Aim 3 will utilize a unique preclinical model in which freshly procured prostate tissue transplanted to humanized mouse hosts as an in vivo model, where prostate tissue architecture is maintained, and all cell compartments, including prostate endothelial cells, are human.